Tumor tissue microarrays for rapid molecular profiling

ABSTRACT

An array-based technology facilitates rapid correlated gene copy number and expression profiling of very large numbers of human tumors. Hundreds of cylindrical tissue biopsies (diameter 0.6 mm) from morphologically representative regions of individual tumors can be arrayed in a single paraffin block. Consecutive sections from such arrays provide targets for parallel in situ visualization and quantitation of DNA, RNA or protein targets. For example, amplifications of six loci (mybL2, erbB2, Cyclin-D1, myc, 17q23 and 20q13) were rapidly determined by fluorescence in situ hybridization from 372 ethanol-fixed breast cancers. Stratification of tumors by estrogen receptor and p53 expression data revealed distinct patterns of gene amplification in the various subgroups of breast cancer that may have prognostic utility. The tissue array technology is useful in the rapid molecular profiling of hundreds of normal and pathological tissue specimens or cultured cells.

PRIORITY CLAIM

This application was filed as a 35 U.S.C. §371 application of PCTInternational Application No. US99/04001 filed Feb. 24, 1999, whichdesignated the United States and was published in English under PCTArticle 21(2), and which in turn claims benefit of U.S. ProvisionalApplication 60/075,979, filed Feb. 25, 1998.

FIELD OF THE INVENTION

The present invention concerns devices for the microscopic, histologicand/or molecular analysis of tissue specimens.

BACKGROUND OF THE INVENTION

Biological mechanisms of many diseases have been clarified bymicroscopic examination of tissue specimens. Histopathologicalexamination has also permitted the development of effective medicaltreatments for a variety of illnesses. In standard anatomical pathology,a diagnosis is made on the basis of cell morphology and stainingcharacteristics. Tumor specimens, for example, can be examined tocharacterize the tumor type and predict whether the patient will respondto a particular form of chemotherapy. Although this microscopicexamination and classification of tumors has improved medical treatment,the microscopic appearance of a tissue specimen stained by standardmethods (such as hematoxylin and eosin) can often only reveal a limitedamount of diagnostic or molecular information.

Recent advances in molecular medicine have provided an even greateropportunity to understand the cellular mechanisms of disease, and selectappropriate treatments with the greatest likelihood of success. Somehormone dependent breast tumor cells, for example, have an increasedexpression of estrogen receptors on their cell surfaces, which indicatesthat the patient from whom the tumor was taken will likely respond tocertain anti-estrogenic drug treatments. Other diagnostic and prognosticcellular changes include the presence of tumor specific cell surfaceantigens (as in melanoma), the production of embryonic proteins (such asα-fetoprotein in liver cancer and carcinoembryonic glycoprotein antigenproduced by gastrointestinal tumors), and genetic abnormalities (such asactivated oncogenes in tumors). A variety of techniques have evolved todetect the presence of these cellular abnormalities, includingimmunophenotyping with monoclonal antibodies, in situ hybridization withprobes, and DNA amplification using the polymerase chain reaction (PCR).

The development of new molecular markers, however, has been impeded bythe inability to group a large number of tissues within a small surfacearea Only a limited amount of hybridoma supernatant may be available,particularly during the early phase of monoclonal antibody generation,which limits the number of specimens that can be analyzed. Even if largequantities of the immunohistologic agent are available, however, thereagents are expensive and may vary in reactivity. These problems ledBattifora et al. to propose in Lab. Invest. 5:244-248 (1986), and inU.S. Pat. No. 4,820,504, that multiple tissue specimens may be groupedtogether on a single slide to enable the specimens to be simultaneouslyscreened by application of a single drop of hybridoma supernatant. Thespecimens were prepared by using a hand-held razor blade to cutdeparaffinized and dehydrated tissue specimens into slices, which werethen bundled together randomly, wrapped in a sausage casing, andre-embedded in paraffin. This technique required a high degree of manualdexterity, and incorporated samples into a composite block in a mannerthat made it difficult to find and identify particular specimens ofinterest.

A modification of this process was disclosed by Wan et al., J. Immunol.Meth. 103:121-129 (1987). and Furmanski et al. in U.S. Pat. No.4,914,022, in which cores of paraffin embedded tissue were obtained fromstandard tissue blocks. The cores were softened and straightened bymanually rolling them on a warm surface and then bundled inside aconventional drinking straw. This method was said to be suitable forsimultaneous histologic testing of multiple tissue specimens, forexample in the characterization of monoclonal antibodies. The techniqueof Miller and Groothuis, A.J.C.P. 96:228-232 (1991) similarly rolledtissue strips into “logs” from which transverse sections were taken tobe embedded in paraffin. The straw and log techniques, however, werelabor intensive, required a high degree of manual dexterity, and alsorandomly arranged the samples in a manner that complicated theidentification of specimens of interest Battifora and Mehta, Lab.Invest. 63:722-724 (1990), and U.S. Pat. No. 5,002.377, attempted toovercome some of the problems of random placement by cutting specimensinto a plurality of narrow strips, which were individually positioned inparallel rectangular grooves in a mold. The tissue strips were embeddedin agar gel that was poured into the grooves to produce a plate-likemember with a series of ridges. Several of the ridged plates werestacked together and embedded in paraffin to form a tissue block. Asimilar approach was proposed by Sundblad, A.J.C.P. 102:192-193 (1993),in which the tissue strips were placed in triangular wedges instead ofrectangular grooves. Slicing the tissue, assembling it into rows, andembedding it in several steps to form the block was a time-consumingmethod that reduced the efficiency of examining a large number of tissuespecimens.

All of these techniques have been inadequate for the efficientpreparation of an array of tissue specimens that can be used for rapidparallel analysis of a variety of independent molecular markers. Thisinefficiency has been a significant problem in fields such as cancerresearch, because cancer development and progression is a multi-stepprocess that involves sequential losses, rearrangements andamplifications of several chromosomal regions and multiple genes. Theseevents lead to a dysregulation of critical signal transduction pathwaysfor cell growth, death, and differentiation. The details of this complexprocess remain incompletely understood, partly because high-throughputstrategies and techniques for analyzing such genetic changes in largenumbers of uncultured human tumors have not been available.

For example, simultaneous analysis of several genes within the same orrelated signal transduction pathways may be necessary to pinpointcritical, rate-limiting steps in the dysregulation of cancer cellgrowth. Furthermore, analysis of structural and numerical changesaffecting several chromosomes, loci and genes at the same time may beneeded to understand the patterns of accumulation of genetic changes indifferent stages of the cancer progression. Finally after novel genesand genetic changes of potential importance in cancer have beenidentified, substantial additional research is usually required todetermine the diagnostic, prognostic and therapeutic significance ofthese molecular markers in clinical oncology.

Since the amount of tissue often becomes rate limiting for such studies,the ability to efficiently procure, fix, store and distribute tissue formolecular analysis in a manner that minimizes consumption of oftenunique, precious tumor specimens is important. It is therefore an objectof this invention to perform large-scale molecular profiling of tissuespecimens (such as tumors) with minimal tissue requirements, in a mannerthat allows rapid parallel analysis of molecular characteristics (suchas gene dosage and expression) from hundreds of morphologicallycontrolled tumor specimens.

SUMMARY OF THE INVENTION

The foregoing objects are achieved by a method of parallel analysis oftissue specimens, in which a plurality of donor specimens are placed inassigned locations in a recipient array, and a plurality of sections areobtained from the recipient array so that each section contains aplurality of donor specimens that maintain their assigned locations. Adifferent histological analysis is performed on each section, todetermine if there are correlations between the results of the differentanalyses at corresponding locations of the array. In particularembodiments, the donor specimen is obtained by boring an elongatedsample, such as a cylindrical core, from donor tissue, and placing thedonor specimen in a receptacle of complementary shape, such as acylindrical core, in the recipient array. Analyses that may be performedon the donor specimens include immunological analysis, nucleic acidhybridization, and clinicopathological characterization of the specimen.

In a more particular embodiment of the method, a recipient block isformed from a rigid embedding medium such as paraffin that can be cutwith a punch or microtome, and a separate donor block is also formed byembedding a biological specimen in the embedding medium. Cylindricalreceptacle cores are bored in the recipient block to form an array ofreceptacles at fixed positions, and cylindrical donor sample cores areobtained from the embedded biological specimen in the donor blocks Thedonor sample cores are then placed in the cylindrical receptacles atassigned locations in the array, and the recipient block is sliced toobtain a cross-section of the donor sample cores in the array, withoutdisrupting the assigned array locations. A different histologicalanalysis may be performed on each section, for example by usingdifferent monoclonal antibodies that recognize distinct antigens, or acombination of antigenically distinct monoclonal antibodies and nucleicacid (e.g. RNA and DNA) probes on sequential sections. The result ofeach distinct histological analysis in each position of the array iscompared, for example to determine if a tissue that expresses anestrogen receptor also has evidence that a particular oncogene has beenactivated.

In a more particular embodiment of the method, a recipient block isformed from a rigid embedding medium such as paraffin that can be cutwith a punch or microtome, and a separate donor block is also formed byembedding a biological specimen in the embedding medium. Cylindricalreceptacle cores are bored in the recipient block to form an array ofreceptacles at fixed positions, and cylindrical donor sample cores areobtained from the embedded biological specimen in the donor block. Thedonor sample cores are then placed in the cylindrical receptacles atassigned locations in the array, and the recipient block is sliced toobtain a cross-section of the donor sample cores in the array, withoutdisrupting the assigned array locations. A different histologicalanalysis may be performed on each section, for example by usingdifferent monoclonal antibodies that recognize distinct antigens, or acombination of antigenically distinct monoclonal antibodies and nucleicacid (e.g. RNA and DNA) probes on sequential sections. The result ofeach distinct histological analysis in each position of the array iscompared, for example to determine if a tissue that expresses anestrogen receptor also has evidence that a particular oncogene has beenactivated. The presence or absence of the estrogen receptor and oncogenecan then be correlated with clinical or pathological information aboutthe tissue (such as the presence of metastatic disease or thehistological grade of a tumor). This simultaneous parallel analysis ofmultiple specimens helps clarify the inter-relationship of multiplemolecular and clinical characteristics of the tissue.

The invention also includes a method of obtaining small elongatedsamples of tissue from a tissue specimen, such as a tumor, andsubjecting the specimen to laboratory analysis, such as histological ormolecular analysis. The elongated tissue sample can be taken from aregion of interest of the tissue specimen, and the size of the sample issmall enough that the characterstic being analyzed is substantiallyhomogenous throughout the small sample. In a disclosed embodiment, thesample is a cylindrical sample punched from the tissue specimen, whereinthe cylindrical specimen is about 14 mm long, and has a diameter ofabout 0.14 mm, for example about 0.3-2.0 mm. In specific embodiments,the cylinder diameter is less than about 1.0 mm, for example 0.6 mm. Thesample is preferably preserved in a manner (such as ethanol fixation)that does not interfere with analysis of nucleic acids, and the samplecan therefore be subjected to any type of molecular analysis, such asany type of molecular analysis based on isolated DNA or RNA.

The invention also includes an apparatus for preparing specimens forparallel analysis of sections of biological material arrays. Theapparatus includes a platform, a tissue donor block on the platform, anda punch that punches or bores a tissue specimen from the donor block.The platform can also carry a recipient block in which the punch formsan array of receptacles at selected positions. Each receptacle can bepositioned so that a tissue specimen can be expelled from the reciprocalpunch into the receptacle. An x-y positioning device incrementally movesthe punch or recipient block with respect to one another as the punchreciprocates, to form the receptacle array. The x-y positioning devicealso aligns sequential receptacles of the recipient block with the punchto deliver tissue specimens from the punch into the receptacle. A styletmay be introduced into the punch to expel the contents of the punch,which may be either paraffin from the recipient block or tissue from thedonor block. Regions of interest of the tissue specimen are located bypositioning a thin section slide over the donor block, to alignstructures of interest in the thin section slide with correspondingtissue specimen regions in the donor block.

The invention also includes a computer implemented system for parallelanalysis of consecutive sections of tissue arrays, in which an x-ypositioning platform moves a tray to a plurality of coordinates thatcorrespond to positions in a recipient block array. A receptacle punchthen punches a receptacle core from a recipient block on the positioningplatform, and a stylet expels the receptacle core from the receptaclepunch. A donor punch (which may be the same or separate from therecipient punch) punches a donor specimen from a donor block on thepositioning platform, and a stylet expels the donor specimen from thedonor punch into the receptacle as the donor punch is introduced intothe receptacle. The donor specimen suitably has a diameter that issubstantially the same as the diameter of the receptacle, so that thedonor specimen fits securely in the receptacle. The computer systemidentifies the tissue by its location in the recipient array, so thatwhen the donor block is sectioned a corresponding position in eachsectional array will contain tissue from the identical donor specimen.

The foregoing and other objects, features, and advantages of theinvention will become more apparent from the following detaileddescription of preferred embodiments which proceeds with reference tothe accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic perspective view of a first embodiment of thepunch device of the present invention, showing alignment of the punchabove a region of interest of donor tissue in a donor block.

FIG. 2 is a view similar to FIG. 1, but in which the punch has beenadvanced to obtain a donor specimen sample.

FIG. 3 is a schematic, perspective view of a recipient black into whichthe donor specimen has been placed.

FIGS. 4-8 illustrate steps in the preparation of thin section arraysfrom the recipient block.

FIG. 9 is a perspective view of a locking device for holding a slidemounted specimen above the tissue in the donor block to locate a regionof interest.

FIG. 10A is a view of an H&E stained, thin section tissue array mountedon a slide for microscopic examination.

FIG. 10B is a magnified view of a portion of the slide in FIG. 10A,showing results of erbB2 mRNA in situ hybridzation on a tissue arrayfrom the region in the small rectangle in FIG. 10A.

FIG. 10C is an electrophoresis gel showing that high molecular weightDNA and RNA can be extracted from the breast cancer specimens.

FIG. 10D is an enlarged view of one of the tissue samples of the arrayin FIG. 10A, showing an immunoperoxidase stain for the erbB2 antigen.

FIG. 10E is a view similar to FIG. 10D, showing high level erbB2 geneamplification detected by fluorescent in situ hybridization (FISH) oftissue in the array by an erbB2 DNA probe.

FIG. 11 is a schematic view illustrating an example of parallel analysisof arrays obtained by the method of the present invention.

FIG. 12 is an enlarged view of a portion of FIG. 11.

FIG. 13 is a top view of a second embodiment of a device for forming thearrays of the present invention.

FIG. 14 is a front view of the device shown in FIG. 13, illustrating theformation of a receptacle in a recipient block with a recipient punch.

FIG. 15 is a view similar to FIG. 14, but showing expulsion of a plugfrom the recipient punch into a discard tray.

FIG. 16 is a view showing a donor punch obtaining a tissue specimen froma donor block.

FIG. 17 is a view showing insertion of the donor tissue into areceptacle of the recipient block.

FIG. 18 is an enlarged view of the donor punch aligned above a structureof interest in the donor block, which is shown in cross-section.

FIG. 19 is an enlarged cross-sectional view of the recipient punch,while

FIG. 20 is a similar view of the donor punch, illustrating the relativecross-sectional diameters of the two punches.

FIG. 21 is a cross-sectional view of the recipient block with the donorspecimens arranged in the recipient array, and with lines of microtomesections of the recipient block being shown.

FIG. 22 is a schematic view of a computer system in which the method ofthe present invention can be implemented.

FIG. 23 is an algorithm illustrating an example of the computerimplemented method of the present invention.

DETAILED DESCRIPTION Embodiment of FIGS. 1-10

A first embodiment of a device for making the microarrays of the presentinvention is shown in FIGS. 1-2, in which a donor block 30 is shown in arectangular container 31 mounted on a stationary platform 32 having anL-shaped edge guide 34 that maintains donor container 31 in apredetermined orientation on platform 32. A punch apparatus 38 ismounted above platform 32, and includes a vertical guide plate 40 and ahorizontal positioning plate 42. The positioning plate 42 is mounted onan x-y stage (not shown) that can be precisely positioned with a pair ofdigital micrometers.

Vertical guide plate 40 has a flat front face that provides a precisionguide surface against which a reciprocal punch base 44 can slide along atrack 46 between a retracted position shown in FIG. 1 and an extendedposition shown in FIG. 2. An elastic band 48 helps control the movementof base 44 along this path, and the limits of advancement and retractionof base 44 are set by track member 46, which forms a stop that limitsthe amplitude of oscillation of base 44. A thin wall stainless steeltube punch 50 with sharpened leading edges is mounted on the flat bottomface of base 44, so that punch 50 can be advanced and retracted withrespect to platform 32, and the container 31 on the platform. The hollowinterior of punch 50 is continuous with a cylindrical bore through base44, and the bore opens at opening Si on a horizontal lip 53 of base 44.

FIG. 1 shows that a thin section of tissue can be obtained from donorblock 30 and mounted on a slide 52 (with appropriate preparation andstaining) so that anatomic and microanatomic structures of interest canbe located in the block 30. Slide 52 can be held above donor block 30 byan articulated arm holder 54 (FIG. 9) with a clamp 56 which securelyholds an edge of a transparent support slide 58. Arm holder 54 canarticulate at joint 60, to swivel between a first position in whichsupport slide 58 is locked in position above container 31, and a secondposition in which support slide 58 moves horizontally out of theposition shown in FIG. 9 to permit free access to punch 50.

In operation, the rectangular container 31 is placed on platform 32(FIG. 1) with edges of container 31 abutting edge guides 34 to holdcontainer 31 in a selected position. A donor block 30 is prepared byembedding a gross tissue specimen (such as a three dimensional tumorspecimen 62) in paraffin. A thin section of donor block 30 is shavedoff, stained, and mounted on slide 52 as thin section 64, and slide 52is then placed on support slide 58 and positioned above donor block 30as shown in FIG. 9. Slide 52 can be moved around on support slide 58until the edges of thin section 64 are aligned with the edges of thegross pathological specimen 62, as shown by the dotted lines in FIG. 9.Arm 54 is then locked in the first position, to which the arm cansubsequently return after displacement to a second position.

A micro-anatomic or histologic structure of interest 66 can then belocated by examining the thin section through a microscope (not shown).If the tissue specimen is, for example, an adenocarcinoma of the breast,then the location of interest 66 may be an area of the specimen in whichthe cellular architecture is suggestive of metaplasia (e.g. pyknoticnuclei, pleomorphism, invasiveness). Once the structure of interest 66is located, the corresponding region of tissue specimen 62 from whichthe thin section structure of interest 66 was obtained is locatedimmediately below the structure of interest 66. As shown in FIG. 1,positioning plate 42 can be moved in the x and y directions (under thecontrol of the digital micrometers or a joystick), or the donor blockcan be moved for larger distances, to align punch 50 in position abovethe region of interest of the donor block 30, and the support slide 58is then horizontally pivoted away from its position above donor block 30around pivot joint 60 (FIG. 9).

Punch 50 is then introduced into the structure of interest in donorblock 30 (FIG. 2) by advancing vertical guide plate 40 along track 46until plate 44 reaches its stop position (which is preset by apparatus38). As punch 50 advances, its sharp leading edge bores a cylindricaltissue specimen out of the donor block 30, and the specimen is retainedwithin the punch as the punch reciprocates back to its retractedposition shown in FIG. 1. The cylindrical tissue specimen cansubsequently be dislodged from punch 50 by advancing a stylet (notshown) into opening 51. The tissue specimen is, for example, dislodgedfrom punch 50 and introduced into a cylindrical receptacle ofcomplementary shape and size in an array of receptacles in a recipientblock 70 shown in FIG. 3.

One or more recipient blocks 70 can be prepared prior to obtaining thetissue specimen from the donor block 30. Block 70 can be prepared byplacing a solid paraffin block in container 31 and using punch 50 tomake cylindrical punches in block 70 in a regular pattern that producesan array of cylindrical receptacles of the type shown in FIG. 3. Theregular array can be generated by positioning punch 50 at a startingpoint above block 70 (for example a corner of the prospective array),advancing and then retracting punch 50 to remove a cylindrical core froma specific coordinate on block 70, then dislodging the core from thepunch by introducing a stylet into opening 51. The punch apparatus orthe recipient block is then moved in a regular increments in the xand/or y directions, to the next coordinate of the array, and thepunching step is repeated. In the specific disclosed embodiment of FIG.3, the cylindrical receptacles of the array have diameters of about 0.6mm, with the centers of the cylinders being spaced by a distance ofabout 0.7 mm (so that there is a distance of about 0.05 mm between theadjacent edges of the receptacles).

In a specific example, core tissue biopsies having a diameter of 0.6 mmand a height of 3-4 mm, were taken from selected representative regionsof individual “donor” paraffin-embedded tumor blocks and preciselyarrayed into a new “recipient” paraffin block (20 mm×45 mm). H&E-stainedsections were positioned above the donor blocks and used to guidesampling from morphologically representative sites in the tumors.Although the diameter of the biopsy punch can be varied, 0.6 mmcylinders have been found to be suitable because they are large enoughto evaluate histological patterns in each element of the tumor array,yet are sufficiently small to cause only minimal damage to the originaldonor tissue blocks, and to isolate reasonably homogenous tissue blocks.Up to 1000 such tissue cylinders can be placed in one 20×45 mm recipientparaffin block. Specific disclosed diameters of the cylinders are0.1-4.0 mm, for example 0.5-2.0 mm, and most specifically less than Imm, for example 0.6 mm. Automation of the procedure, with computerguided placement of the specimens, allows very small specimens to beplaced tightly together in the recipient array.

FIG. 4 shows the array in the recipient block after the receptacles ofthe array have been filled with tissue specimen cylinders. The topsurface of the recipient block is then covered with an adhesive film 74from an adhesive coated tape sectioning system (Instrumedics) to helpmaintain the tissue cylinder sections in place in the array once it iscut With the adhesive film in place, a 4-8 μm section of the recipientblock is cut transverse to the longitudinal axis of the tissue cylinders(FIG. 5) to produce a thin microarray section 76 (containing tissuespecimen cylinder sections in the form of disks) that is transferred toa conventional specimen slide 78. The microarray section 76 is adheredto slide 78, for example by adhesive on the slide. The film 74 is thenpeeled away from the underlying microarray member 76 to expose it forprocessing. A darkened edge 80 of slide 78 is suitable for labeling orhandling the slide.

A breast cancer tissue specimen was fixed in cold ethanol to helppreserve high-molecular weight DNA and RNA, and 372 of the specimenswere fixed in this manner. At least 200 consecutive 4-8 μm tumor arraysections can be cut from each block providing targets for correlated insitu analyses of copy number or expression of multiple genes. Thisanalysis is performed by testing for different gene amplifications inseparate array sections, and comparing the results of the tests atidentical coordinates of the array (which correspond to tissue specimensfrom the same tissue cylinder obtained from donor block). This approachenables measurement of virtually hundreds of molecular characteristicsfrom every tumor, thereby facilitating construction of a large series ofcorrelated genotypic or phenotypic characteristics of uncultured humantumors.

An example of a single microarray 76 containing 645 specimens is shownin FIG. 10A. An enlarged section of the microarray (highlighted by arectangle in FIG. 10A) is shown in FIG. 10B, in which an autoradiogramof erbB2 mRNA in situ hybridization illustrates that two adjacentspecimens in the array demonstrate a strong hybridization signal. FIG.10C illustrates electrophoresis gels which demonstrate that highmolecular weight DNA and RNA can be extracted from breast cancerspecimens fixed in ethanol at 4° C. overnight in a vacuum oven.

One of the tissue specimens that gave the fluorescent “positive” signalswas also analyzed by immunoperoxidase staining, as shown in FIG. 10D,where it was confirmed (by the dark stain) that the erbB2 gene productwas present. A DNA probe for the erbB2 gene was used to performfluorescent in situ hybridization (FISH). FIG. 10D shows one of thetumor array elements, which demonstrated high level erbB2 geneamplification. The insert in FIG. 10E shows three nuclei with numeroustightly clustered erbB2 hybridization signals and two copies of thecentromeric reference probe. Additional details about these assays aregiven in Examples 1-4 below.

The potential of the array technology of the present invention toperform rapid parallel molecular analysis of multiple tissue specimensis illustrated in FIG. 11, where the y-axis of the graphs corresponds topercentages of tumors in specific groups that have definedclinicopathological or molecular characteristics. This diagram showscorrelations between clinical and histopathological characteristics ofthe tissue specimens in the micro-array. Each small box in the alignedrows of FIG. 11B represents a coordinate location in the array.Corresponding coordinates of consecutive thin sections of the recipientblock are vertically aligned above one another in the horizontallyextending rows. These results show that the tissue specimens could beclassified into four classifications of tumors (FIG. 11A) based on thepresence or absence of cell membrane estrogen receptor expression, andthe presence or absence of the p53 mutation in the cellular DNA. In FIG.11B, the presence of the p53 mutation is shown by a darkened box, whilethe presence of estrogen receptors is also shown by a darkened box.Categorization into each of four groups (ER−/p53+, ER−/p53−, ER+/p53+and ER+/p53−) is shown by the dotted lines between FIGS. 11A and 11B,which divide the categories into Groups I, II, III and IV correspondingto the ER/p53 status.

FIG. 11B also shows clinical characteristics that were associated withthe tissue at each respective coordinate of the array. A darkened boxfor Age indicates that the patient is premenopausal, a darkened box Nindicates the presence of metastatic disease in the regional lymphnodes, a darkened box T indicates a stage 3 or 4 tumor which is moreclinically advanced, and a darkened box for grade indicates a high grade(at least grade III) tumor, which is associated with increasedmalignancy. The correlation of ER/p53 status can be performed bycomparing the top four lines of clinical indicator boxes (Age, N, T,Grade) with the middle two lines of boxes (ER/p53 status). The resultsof this cross correlation are shown in the bar graph of FIG. 11A, whereit can be seen that ER−/p53+ (Group I) tumors tend to be of higher gradethan the other tumors, and had a particularly high frequency of mycamplification, while ER+/p53+ (Group III) tumors were more likely tohave positive nodes at the time of surgical resection. The ER−/p53−(Group II) showed that the most common gene amplified in that group waserbB2. ER−/p53− (Group II) and ER+/p53− (Group IV) tumors, in contrast,were shown to have fewer indicators of severe disease, thus suggesting acorrelation between the absence of the p53 mutation and a betterprognosis.

This method was also used to analyze the copy numbers of several othermajor breast cancer oncogenes in the 372 arrayed primary breast cancerspecimens in consecutive FISH experiments, and those results were usedto ascertain correlations between the ER/p53 classifications and theexpression of these other oncogenes. These results were obtained byusing probes for each of the separate oncogenes, in successive sectionsof the recipient block. and comparing the results at correspondingcoordinates of the array. In FIG. 11B, a positive result for theamplification of the specific oncogene or marker (mybL2, 20q13, 17q23,myc, cnd1 and erbB2) is indicated by a darkened box. The erbB2 oncogenewas amplified in 18% of the 372 arrayed specimens, myc in 25% and cyclinD1 (cnd1) in 24% of the tumors.

The two recently discovered novel regions of frequent DNA amplificationin breast cancer, 17q23 and 20q13, were found to be amplified in 13% and6% of the tumors, respectively. The oncogene mybL2 (which was recentlylocalized to 20q13.1 and found to be overexpressed in breast cancer celllines) was found to be amplified in 7% of the same set of tumors. MybL2was amplified in tumors with normal copy number of the main 20q13 locus,indicating that it may define an independently selected region ofamplification at 20q. Dotted lines between FIGS. 11B and 11C againdivide the complex co-amplification patterns of these genes into GroupsI-IV which correspond to ER−/p53+, ER−/p53−, ER+/p53+ and ER+/p53−.

FIGS. 11C and 11D show that 70% of the ER−/p53+ specimens were positivefor one or more of these oncogenes, and that myc was the predominantoncogene amplified in this group. In contrast, only 43% of the specimensin the ER+/p53− group showed co-amplification of one of these oncogenes,and this information could in turn be correlated with the clinicalparameters shown in FIG. 11A. Hence the microarray technology permits alarge number of tumor specimens to be conveniently and rapidly screenedfor these many characteristics, and analyzed for patterns of geneexpression that may be related to the clinical presentation of thepatient and the molecular evolution of the disease. In the absence ofthe microarray technology of the present invention, these correlationsare more difficult to obtain.

A specific method of obtaining these correlations is illustrated in FIG.12, which is an enlargement of the right hand portion of FIG. 11B. Themicroarray 76 (FIG. 10A) is arranged in sections that contain seventeenrows and nine columns of circular locations that correspond tocross-sections of cylindrical tissue specimens from different tumors,wherein each location in the microarray can be represented by thecoordinates (row, column). For example, the specimens in the first rowof the first section have coordinate positions (1,1), (1,2) . . . (1,9),and the specimens in the second row have coordinate positions (2,1),(2,2) . . . (2,9). Each of these array coordinates can be used to locatetissue specimens from corresponding positions on sequential sections ofthe recipient block, to identify tissue specimens of the array that werecut from the same tissue cylinder.

As shown in FIG. 12, the rectangular array is converted into a linearrepresentation in which each box of the linear representationcorresponds to a coordinate position of the array. Each of the lines ofboxes is aligned so that each box that corresponds to an identical arraycoordinate position is located above other boxes from the samecoordinate position. Hence the boxes connected by dotted line 1correspond to the results that can be obtained by looking at the resultsat coordinate position (1,1) in successive thin sections of the donorblock, or clinical data that may not have been obtained from themicroarray, but which can be entered into the system to further identifytissue from a tumor that corresponds to that coordinate position.Similarly, the boxes connected by dotted line 10 correspond to theresults that can be found at coordinate position (2,1) of the array, andthe boxes connected by dotted line 15 correspond to the results atcoordinate position (2,6) of the array. The letters a, b, c, d, e, f, g,and h correspond to successive sections of the donor block that are cutto form the array.

By comparing the aligned boxes along line 1 in FIG. 12, it can be seenthat a tumor was obtained from a postmenopausal woman with no metastaticdisease in her lymph nodes at the time of surgical resection, in whichthe tumor was less than stage 3, but in which the histology of the tumorwas at least Grade III. A tissue block was taken from this tumor andintroduced into the recipient array at coordinate position (1,1), andonce the array was completed it was sectioned into eight parallelsections (a, b, c, d, e, f, g, and h) each of which contained arepresentative section of the cylindrical array. Each of these sectionswas analyzed with a different probe specific for a particular molecularattribute. In section a, the results indicated that this tissue specimenwas p53+; in section b that it was ER−; in section c that it did notshow amplification of the mybL2 oncogene; in separate sections d, e, f,g and h that it was positive for the amplification of 20q13, 17q23, myc,cnd1 and erbB2.

Similar comparisons of molecular characteristics of the tumor specimencylinder that was placed at coordinate position (2,1) can be made byfollowing vertical line 10 in FIG. 12, which connects the tenth box ineach line, and corresponds to the second row, first column (2,1) of thearray 76 in FIG. 10(A). Similarly the characteristics of the sections ofthe tumor specimen cylinder at coordinate position (2,6) can be analyzedby following vertical line 15 down through the 15^(th) box of each row.In this manner, parallel information about the separate sections of thearray can be performed for all 372 positions of the array. Thisinformation can be presented visually for analysis as in FIG. 12, orentered into a database for analysis and correlation of differentmolecular characteristics (such as patterns of oncogene amplification,and the correspondence of those patterns of amplification to clinicalpresentation of the tumor).

Analysis of consecutive sections from the arrays enables co-localizationof hundreds of different DNA, RNA or protein targets in the same cellpopulations in morphologically defined regions of every tumor, whichfacilitates construction of a database of a large number of correlatedgenotypic or phenotypic characteristics of uncultured human tumors.Scoring of mRNA in situ hybridizations or protein immunohistochemicalstaining is also facilitated with tumor tissue microarrays, becausesmall amounts of the identical reagents are used for each analysis. Thetumor arrays also substantially reduce tissue consumption, reagent use,and workload when compared with processing individual conventionalspecimens for sectioning, staining and scoring. The combined analysis ofseveral DNA, RNA and protein targets provides a powerful means forstratification of tumor specimens by virtue of their molecularcharacteristics. Such patterns will be helpful to detect previouslyunappreciated but important molecular features of the tumors that mayturn out to have diagnostic or prognostic utility.

These results show that the very small cylinders used to prepare tissuearrays can in most cases provide accurate information, especially whenthe site for tissue sampling from the donor block is selected to containhistological structures that are most representative of tumor regions.It is also possible to collect samples from multiple histologicallydefined regions in a single donor tissue block to obtain a morecomprehensive representation of the original tissue, and to directlyanalyze the correlation between phenotype (tissue morphology) andgenotype. For example, an array could be constructed to include hundredsof tissues representing different stages of breast cancer progression(e.g. normal tissue, hyperplasia, atypical hyperplasia, intraductalcancer, invasive and metastatic cancer). The tissue array technologywould then be used to analyze the molecular events that correspond totumor progression.

A tighter packing of cylinders and a larger recipient block can alsoprovide an even higher number of specimens per array. Entire archivesfrom pathology laboratories could be placed in replicate 1000 specimentissue microarrays for molecular profiling. Using automation of theprocedure for sampling and arraying, it is possible to make dozens ofreplicate tumor arrays, each providing hundreds of sections formolecular analyses. The same strategy and instrumentation developed fortumor arrays also enables microdissection of tissue cylinders forisolation of high-molecular weight RNA and DNA from optimally fixed,morphologically defined tumor tissue elements, thereby allowingcorrelated analysis of the same tumors by PCR-based techniques for RNAand DNA. When nucleic acid analysis is planned, the tissue specimen ispreferably fixed (before embedding in paraffin) in ethanol or MolecularBiology Fixative (Streck Laboratories, Inc., Omaha, Nebr.) instead of informalin, because formalin can cross-link and otherwise damage nucleicacid. The tissue cylinder of the present invention provides an ampleamount of DNA or RNA on which to perform a variety of molecularanalyses.

The potential of this array technology has been illustrated in FISHanalysis of gene amplifications in breast cancer. FISH is an excellentmethod for visualization and accurate detection of geneticrearrangements (amplifications, deletions or translocations) inindividual, morphologically defined cells. The combined tumor arraytechnology allows FISH to become a powerful, high-throughput method thatpermits the analysis of hundreds of specimens per day.

Embodiment of FIGS. 13-23

An example of an automated system for high speed preparation of themicroarrays is shown in FIGS. 13-23. The system includes a stage 100having an x drive 102 and a y drive 104, each of which respectivelyrotates a drive shaft 106, 108. The shaft 108 moves a specimen bench 110in a y direction, while the shaft 106 moves a tray 112 on the bench 110in an x direction. Mounted in a front row of tray 112 are threerecipient containers 116, 118 and 120, each of which contains arecipient paraffin block 122, 124 or 126, and a donor container 128 thatcontains a donor paraffin block 130, in which is embedded a tissuespecimen 132. In a back row on the tray are two multi-well donor trays132, 134 (which contain multiple containers for maintaining specimens inliquid medium), and a discard container 136.

Disposed above stage 100 is a punch apparatus 140 that can move up anddown in a z direction. Apparatus 140 includes a central, verticallydisposed, stylet drive 142 in which reciprocates a stylet 144. Apparatus140 also includes an inclined recipient punch drive 146, and a inclineddonor punch drive 148. Punch drive 146 includes a reciprocal ram 150that carries a tubular recipient punch 154 at its distal end, and punchdrive 148 includes a reciprocal ram 152 that carries a donor tubularpunch 156 at its distal end. When the ram 150 is extended (FIG. 14),recipient punch 154 is positioned with the open top of its tubular borealigned with stylet 144, and when ram 152 is extended (FIG. 16), donorpunch 156 is positioned with the open top of its tubular bore alignedwith stylet 144.

The sequential operation of the apparatus 140 is shown in FIGS. 13-17.Once the device is assembled as in FIG. 13, a computer system can beused to operate the apparatus to achieve high efficiency. Hence thecomputer system can initialize itself by determining the location of thecontainers on tray 112 shown in FIG. 13. The x and y drives 102, 104 arethen activated to move bench 110 and tray 112 to the position shown inFIG. 14, so that activation of ram 150 extends recipient punch 154 to aposition above position (1,1) in the recipient block 122. Once punch 154is in position, apparatus moves downward in the z direction to punch acylindrical bore in the paraffin of the recipient block. The apparatus140 then moves upwardly in the z direction to raise punch 154 out of theparaffin recipient block 122, but the punch 154 retains a core ofparaffin that leaves a cylindrical receptacle in the recipient block122. The x-y drives are then activated to move bench 110 and positiondiscard container 136 below punch 154. Stylet drive 142 is thenactivated to advance stylet 144 into the open top of the aligned punch154, to dislodge the paraffin core from punch 154 and into discardcontainer 136.

Stylet 144 is retracted from recipient punch 154, ram 150 is retracted,and the x-y drive moves bench 110 and tray 112 to place donor container128 is a position (shown in FIG. 16) such that advancement of ram 152advances donor punch 156 to a desired location over the donor block 130.Apparatus 140 is then moved down in the z direction to punch acylindrical core of tissue out of the donor block 130, and apparatus 140is then moved in the z direction to withdraw donor punch 156, with thecylindrical tissue specimen retained in the punch. The x-y drive thenmoves bench 110 and tray 112 to the position shown in FIG. 17, such thatmovement of apparatus 140 downwardly in the z direction advances donorpunch 156 into the receptacle at the coordinate position (1,1) in block122 from which the recipient plug has been removed. Donor punch 156 isaligned below stylet 144, and the stylet is advanced to dislodge theretained tissue cylinder from donor punch 156, so that the donor tissuecylinder remains in the receptacle of the recipient block 122 as theapparatus 140 moves up in the z direction to retract donor punch 156from the recipient array. Ram 152 is then retracted.

This process can be repeated until a desired number of recipientreceptacles have been formed and filled with cylindrical donor tissuesat the desired coordinate locations of the array. Although thisillustrated method shows sequential alternating formation of eachreceptacle, and introduction of the tissue cylinder into the formedreceptacle, it is also possible to form all the receptacles in recipientblocks 122, 124 and 126 as an initial step, and then move to the step ofobtaining the tissue specimens and introducing them into the preformedreceptacles. The same tissue specimen 132 can be repeatedly used, or thespecimen 132 can be changed after each donor tissue specimen isobtained, by introducing a new donor block 130 into container 128. Ifthe donor block 130 is changed after each tissue cylinder is obtained,each coordinate of the array can include tissue from a different tissuespecimen.

A positioning device is shown in FIG. 18, which helps locate structuresof interest from which donor specimens can be taken. The positioningdevice includes a support slide 160 that extends between opposing wallsof donor container 128, to support a specimen slide 162 on which ismounted a thin stained section of the specimen 132 in donor block 130.Using a microscope mounted on apparatus 140 (the objective of themicroscope is shown at 166), microanatomic structures of interest can befound. The correct vertical height of apparatus 140 above the topsurface of donor block 130 can be determined by the use of twopositioning lights 168, 170 that am mounted to apparatus 140. Lightbeams 172, 174 are projected from lights 168, 170 at an angle such thatthe beams coincide at a single spot 176 when vertical height ofapparatus 140 above the top surface of the light is at a desired zlevel. This desired z level will position the punches 152, 154 at anappropriate height to penetrate the surface of block 130 at the desiredlocation, and to a desired depth.

It is advantageous if the tissue cylinders punched from block 130 fitsecurely in the recipient receptacles that are formed to receive them.If the donor punch 156 has the same inner and outer diameters as therecipient punch 154, then the cylindrical donor tissue specimen will beformed by the inner diameter of the punch, and the recipient receptaclewill be formed by the outer diameter of the punch. This discrepancy willprovide a receptacle that is slightly larger in diameter than the donortissue cylinder. Hence, as shown in FIGS. 19 and 20, the recipient punch154 preferably has a smaller diameter than the donor punch 156.Recipient punch will therefore form a cylindrical receptacle (having adiameter corresponding to the outer diameter of punch 154) that issubstantially the same diameter as the tissue specimen cylinder 180,which is formed with a diameter that is determined by the inner diameterof the donor punch 156.

FIG. 21 illustrates a cross-section through the recipient array, oncethe receptacles 182 have been formed and filled with tissue specimencylinders 180. Small partitions of paraffin material 122 separate tissuecylinders 18, and the receptacles 182 as illustrated are deeper than thespecimen cylinders 180, such that a small clearance is present betweenthe specimen and the bottom of the receptacles. Once the array has beenformed, a microtome can be used to cut a thin section S off the top ofthe block 122, so that the section S can be mounted on a specimen slide162 (FIG. 18) to help locate structures of interest in the tissuespecimen 132. The microtome then also cuts thin parallel sections a, b,c, d, e, f, g, and h that can each be subjected to a different molecularanalysis, as already described.

Exemplary Operating Environment

FIG. 22 and the following discussion are intended to provide a brief,general description of a suitable computing environment in which theinvention may be implemented. The invention is implemented in a varietyof program modules. Generally, program modules include routines,programs, components, data structures, ect. that perform particulartasks or implement particular abstract data types. The invention may bepracticed with other computer system configurations, including hand-helddevices, multiprocessor systems, microprocessor-based or programmableconsumer electronics, minicomputers, mainframe computers, and the like.The invention may also be practiced in distributed computingenvironments where tasks are performed by remote processing devices thatare linked through a communications network. In a distributed computingenvironment, program modules may be located in both local and remotememory storage devices.

Referring to FIG. 22, an operating environment for an illustratedembodiment of the present invention is a computer system 220 with acomputer 222 that comprises at least one high speed processing unit(CPU) 224, in conjunction with a memory system 226, an input device 228,and an output device 230. These elements are interconnected by at leastone bus structure 232.

The illustrated CPU 224 is of familiar design and includes an ALU 234for performing computations, a collection of registers 236 for temporarystorage of data and instructions, and a control unit 238 for controllingoperation of the system 220. The CPU 224 may be a processor having anyof a variety of architectures including Alpha from Digital; MIPS fromMIPS Technology, NEC, IDT, Siemens and others; x 86 from Intel andothers, including Cyrix, AMD, and Nexgen; 680 x 0 from Motorola; andPowerPC from IBM and Motorola.

The memory system 226 generally includes high-speed main memory 240 inthe form of a medium such as random access memory (RAM) and read onlymemory (ROM) semiconductor devices, and secondary storage 242 in theform of long term storage mediums such as floppy disks, hard disks,tape, CD-ROM, flash memory, etc. and other devices that store data usingelectrical, magnetic, optical or other recording media. The main memory240 also can include video display memory for displaying images througha display device. Those skilled in the art will recognize that thememory 226 can comprise a variety of alternative components having avariety of storage capacities.

The input and output devices 228, 230 also are familiar. The inputdevice 228 can comprise a keyboard, a mouse, a scanner, a camera, acapture card, a limit switch (such as home, safety or state switches), aphysical transducer (e.g., a microphone), etc. The output device 230 cancomprise a display, a printer, a motor driver, a solenois, a transducer(e.g., a speaker), etc. Some devices, such as a network interface or amodem, can be used as input and/or output devices.

As is familiar to those skilled in the art, the computer system 220further includes an operating system and at least one applicationprogram. The operating system is the set of software which controls thecomputer system's operation and the allocation of resources. Theapplication program is the set of software that performs a task desiredby the user, using computer resources made available through theoperating system. Both are resident in the illustrated memory system226.

For example, the invention could be implemented with a Power Macintosh8500 available from Apple Computer, or an IBM compatible PersonalComputer (PC). The Power Mcintosh uses a PowerPC 604 CPU from Motorolaand runs a MacOS operating system from Apple Computer such as System 8.Input and output devices can be interfaced with the CPU using thewell-known SCSI interface or with expansion cards using the PeripheralComponent Interconnect (PCI) bus. A typical configuration of a PowerMacintosh 8500 has 72 megabytes of RAM for high-speed main memory and a2 gigabyte hard disk for secondary storage. An IBM compatible PC couldhave a configuration with 32 megabytes of RAM for high-speed main memoryand a 2-4 gigabyte hard disk for secondary storage.

In accordance with the practices of persons skilled in the art ofcomputer programming, the present invention is described with referenceto acts and symbolic representations of operations that are performed bythe computer system 220, unless indicated otherwise. Such acts andoperations are sometimes referred to as being computer-executed. It willbe appreciated that the acts and symbolically represented operationsinclude the manipulation by the CPU 224 of electrical signalsrepresenting data bits which causes a resulting transformation orreduction of the electrical signal representation, and the maintenanceof data bits at memory locations in the memory system 226 to therebyreconfigure or otherwise alter the computer system's operation, as wellas other processing of signals. The memory locations where data bits aremaintained are physical locations that have particular electrical,magnetic, or optical properties corresponding to the data bits.

Description or Computer-Array System

A block diagram showing a system for carrying out the invention is shownat FIG. 23. The hardware is initialized at step 250, for example bydetermining the position of the punches 154, 156, bench 110, and tray112. The system may then be configured by the operator at step 252, forexample by entering data or prompting the system to find the location(x, y, z coordinates) of the upper right corner of each recipient block122-126, as well as the locations of trays 130-136. The number of donorblocks, receptacles, operating speed, etc. may also be entered at thistime.

At step 254, the system prompts for entry of identifying informationabout the first donor block 130 that will be placed in tray 128. Thisidentifying information can include accession number information,clinical information about the specimen, and any/or other informationthat would be useful in analyzing the tumor arrays. At step 256, theoperator pushes a select function button, which raises the punches 154,156 and enables a joystick to move the specimens using the x-y drives.The entered data is displayed at step 258, and approved at 260.

The system then obtains one or more donor specimens from the identifieddonor block at step 262, and prompts the user for entry of informationabout the next donor block. If information about another block isentered, the system returns to step 256 and obtains the desired numberof specimens from the new block. After a new donor block has been placedin donor container 128, the system also checks the position of thepunches at step 268. If information about another block is not enteredat step 264, the system moves the donor tray to the reloading positionto that a block 130 in the donor tray can be removed. This system isalso adaptable to sampling cylindrical biopsies from histologicallycontrolled sites of specimens (such as tumors) for DNA/RNA isolation.

The automated tumor array technology easily allows testing of dozens orhundreds of markers from the same set of tumors. These studies can becarried out in a multi-center setting by sending replicate tumor arrayblocks or sections to other laboratories. The same approach would beparticularly valuable for testing newly discovered molecular markers fortheir diagnostic, prognostic or therapeutic utility. The tissue arraytechnology also facilitates basic cancer research by providing aplatform for rapid profiling of hundreds or thousands of tumors at theDNA, RNA and protein levels, leading to a construction of a correlateddatabase of biomarkers from a large collection of tumors. For example,search for amplification target genes requires correlated analyses ofamplification and expression of dozens of candidate genes and loci inthe same cell populations. Such extensive molecular analyses of adefined large series of tumors would be difficult to carry out withconventional technologies.

Examples of Array Technology

Applications of the tissue array technology are not limited to studiesof cancer, although the following Examples 1-4 disclose embodiments ofits use in connection with analysis of neoplasms. Array analysis couldalso be instrumental in understanding expression and dosage of multiplegenes in other diseases, as well as in normal human or animal tissues,including repositories of tissues from different transgenic animals orcultured cells. The following specific examples illustrate someparticular embodiments of the invention.

EXAMPLE 1 Tissue Specimens

A total of 645 breast cancer specimens were used for construction of abreast cancer tumor tissue microarray. The samples included 372fresh-frozen ethanol-fixed tumors, as well as 273 formalin-fixed breastcancers, normal tissues and fixation controls. The subset of frozenbreast cancer samples was selected at random from the tumor bank of theinstitute of Pathology, University of Basel, which includes more than1500 frozen breast cancers obtained by surgical resections during1986-1997. Only the tumors from this tumor bank were used for molecularanalyses. This subset was reviewed by a pathologist, who determined thatthe specimens included 259 ductal, 52 lobular, 9 medullary, 6 mucinous,3 cribriform, 3 tubular, 2 papillary, 1 histiocytic, 1 clear cell, and 1lipid rich carcinoma. There were also 15 ductal carcinomas in situ, 2carcinosarcomas, 4 primary carcinomas that had received chemotherapybefore surgery, 8 recurrent tumors and 6 metastases. Histologicalgrading was only performed in invasive primary tumors that had notundergone previous chemotherapy. Of these tumors, 24% were grade 1,40%/a grade 2, and 36% grade 3. The pT stage was pT1 in 29%, pT2 in 54%,pT3 in 9%, and pT4 in 8%. Axillary lymph nodes had been examined in 282patients (45% pN0, 46% pN1, 9% pN2). All previously unfixed tumors werefixed in cold ethanol at +4° C. overnight and then embedded in paraffin.

EXAMPLE 2 Immunohistochemistry

After formation of the array and sectioning of the donor block, standardindirect immunoperoxidase procedures were used for immunohistochemistry(ABC-Elite, Vector Laboratories). Monoclonal antibodies from DAKO(Glostrup, Denmark) were used for detection of p53 (DO-7, mouse, 1:200),erbB-2 (c-erbB-2, rabbit, 1:4000), and estrogen receptor (ER IDS, mouse,1:400). A microwave pretreatment was performed for p53 (30 minutes at90°) and erbB-2 antigen (60 minutes at 90°) retrieval. Diaminobenzidinewas used as a chromogen. Tumors with known positivity were used aspositive controls. The primary antibody was omitted for negativecontrols. Tumors were considered positive for ER or p53 if anunequivocal nuclear positivity was seen in at least 10% of tumor cells.The erbB-2 staining was subjectively graded into 3 groups: negative (nostaining), weakly positive (weak membranous positivity), stronglypositive (strong membranous positivity).

EXAMPLE 3 Fluorescent In Situ Hybridization (FISH)

Two-color FISH hybridizations were performed using Spectrumm-Orangelabeled cyclin D1, myc or erbB2 probes together with corresponding FITClabeled centromeric reference probes (Vysis). One-color FISHhybridizations were done with spectrum orange-labeled 20q13 minimalcommon region (Vysis, and see Tanner et al., Cancer Res. 54:4257-4260(1994)), mybL2 and 17q23 probes (Barlund et al., Genes Chrom. Cancer20:372-376 (1997)). Before hybridization, tumor array sections weredeparaffinized, air dried and dehydrated in 70, 85 and 100% ethanolfollowed by denaturation for 5 minutes at 74° C. in 70% formamide-2×SSCsolution. The hybridization mixture contained 30 ng of each of theprobes and 15 μg of human Cot1-DNA. After overnight hybridization at 37°C. in a humidified chamber, slides were washed and counterstained with0.2 μM DAPI in an antifade solution. FISH signals were scored with aZeiss fluorescence microscope equipped with double-band pass filters forsimultaneous visualization of FITC and Spectrum Orange signals. Over 10FISH signals per cell or tight clusters of signals were considered ascriteria for gene amplification.

EXAMPLE 4 mRNA In Situ Hybridization

For mRNA in situ hybridization, tumor array sections were deparaffinizedand air dried before hybridization. Synthetic oligonucleotide probesdirected against erbB2 mRNA (Genbank accession number X03363,nucleotides 350-396) was labeled at the 3′-end with ³³P-dATP usingterminal deoxynucleotidyl transferase. Sections were hybridized in ahumidified chamber at 42° C. for 18 hours with 1×10⁷ CPM/ml of the probein 100 μL of hybridization mixture (50% formamide, 10% dextran sulfate,1% sarkosyl, 0.02 M sodium phosphate, pH 7.0, 4×SSC, 1×Denhardfssolution and 10 mg/ml ssDNA). After hybridization, sections were washedseveral times in 1×SSC at 55° C. to remove unbound probe, and brieflydehydrated. Sections were exposed for three days to phosphorimagerscreens to visualize ERBB2 mRNA expression. Negative control sectionswere treated with RNase prior to hybridization, which abolished allhybridization signals.

The present method enables high throughput analysis of hundreds ofspecimens per array. This technology therefore provides an order ofmagnitude increase in the number of specimens that can be analyzed, ascompared to prior blocks where a few dozen individual formalin-fixedspecimens are in a less defined or undefined configuration, and used forantibody testing. Further advantages of the present invention includenegligible destruction of the original tissue blocks, and an optimizedfixation protocol which expands the utility of this technique tovisualization of DNA and RNA targets. The present method also permitsimproved procurement and distribution of human tumor tissues forresearch purposes. Automation of the procedure permits efficientspecimen sampling and array formation into multiple tissue arrays, eachproviding as many as 50, 100 or even up to 200 sections for molecularanalysis. Entire archives of tens of thousands of existingformalin-fixed tissues from pathology laboratories can be placed in afew dozen high-density tissue microarrays to survey many kinds of tumortypes, as well as different stages of tumor progression. The tumor arraystrategy also allows testing of dozens or even hundreds of potentialprognostic or diagnostic molecular markers from the same set of tumors.Alternatively, the cylindrical tissue samples provide specimens that canbe used to isolate DNA and RNA for molecular analysis.

In view of the many possible embodiments to which the principles of ourinvention may be applied, it should be recognized that the illustratedembodiments are preferred examples of the invention, and should not betaken as a limitation on the scope of the invention. Rather, the scopeof the invention is defined by the following claims. We therefore claimas our invention all that comes within the scope and spirit of theseclaims.

We claim:
 1. An apparatus for preparing specimens for parallel analysisof sections of biological material arrays, comprising: a donor blockholder for holding a tissue donor block in a donor position; and a firstreciprocating punch positioned in relation to the holder to punch atissue specimen from the tissue donor block when the donor block is inthe donor position; a recipient block holder for holding a recipientblock in a recipient position, wherein the recipient block comprises anarray of receptacles, each of which is positionable in a preselectedposition in relation to the first reciprocating punch to deliver atissue specimen from the first reciprocating punch into a receptacle inthe preselected position; and a second reciprocating punch capable ofbeing positioned relative to the recipient block for punching the arrayof receptacles in the recipient block, wherein the second reciprocatingpunch is different than the first reciprocating punch positioned topunch the specimen from the tissue donor block; and a positioner forpositioning over the donor block a reference slide that includes atleast one structure of interest, to align the at least one structure ofinterest in the reference slide with corresponding tissue specimenregions in the donor block.
 2. The apparatus of claim 1, wherein therecipient block bolder comprises an x-y positioning device that can beincrementally moved to align sequential receptacles and thereciprocating punch.
 3. The apparatus of claim 1, further comprising astylet positioned for introduction into the reciprocating punch to expelthe tissue specimen from the punch into one of the receptacles alignedwith the punch.
 4. The apparatus of claim 1, wherein the diameter of thefirst reciprocating punch positioned to punch the specimen from thetissue donor block is greater than the diameter of the secondreciprocating punch.
 5. The apparatus of claim 1, further comprising amicroscope configured for observing the reference slide.
 6. An apparatusfor preparing specimens for parallel analysis of sections of biologicalmaterial arrays, comprising: a donor block holder for holding a tissuedonor block in a donor position; and a first reciprocating punchpositioned in relation to the holder to punch a tissue specimen from thetissue donor block when the donor block is in the donor position; and arecipient block holder for holding a recipient block in a recipientposition, wherein the recipient block comprises an array of receptacles,each of which is positionable in a preselected position in relation tothe first reciprocating punch to deliver a tissue specimen from thefirst reciprocating punch into a receptacle in the preselected position;and a second reciprocating punch capable of being positioned relative tothe recipient block for punching the array of receptacles in therecipient block, wherein the second reciprocating punch is differentthan the fist reciprocating lunch positioned to punch the specimen fromthe tissue donor block; and a recorder for recording coordinatepositions of the receptacles in the recipient block.
 7. The apparatus ofclaim 6, wherein the recorder is a computer implemented system forrecording the positions of the receptacles, and recording anidentification of the tissue specimen that is placed in each receptacle.8. The apparatus of claim 7 wherein the identification includesinformation about the biological material that is not obtained fromanalysis of sections of the biological material.
 9. The apparatus ofclaim 6, wherein the diameter of the first reciprocating punchpositioned to punch the specimen from the tissue donor block is greaterthan the diameter of the second reciprocating punch.
 10. An apparatusfor preparing specimens for parallel analysis of sections of biologicalmaterial arrays, comprising: a donor block holder for holding a tissuedonor block in a donor position; and a first reciprocating punchpositioned in relation to the holder to punch a tissue specimen from thetissue donor block when the donor block is in the donor position; and arecipient block holder for holding a recipient block in a recipientposition, wherein the recipient block comprises an array of receptacles,each of which is positionable in a preselected position in relation tothe first reciprocating punch to deliver a tissue specimen from thefirst reciprocating punch into a receptacle in the preselected position;and a second reciprocating punch capable of being positioned relative tothe recipient block for punching the array of receptacles in therecipient block, wherein the second reciprocating punch is differentthan the first reciprocating punch positioned to punch the specimen fromthe tissue donor block; and a sectioning device for sectioning therecipient block into sections that can be subjected to differentanalyses.
 11. The apparatus of claim 10, further comprising a recorderfor recording results of the different analyses in association withinformation about the biological material that is not obtained fromanalysis of the sections themselves.
 12. The apparatus of claim 10,wherein the diameter of the first reciprocating punch positioned topunch the specimen from the tissue donor block is greater than thediameter of the second reciprocal punch.
 13. An apparatus for preparingspecimens for parallel analysis of sections of biological materialarrays, comprising: a donor block holder for holding a tissue donorblock in a donor position; and a first reciprocating punch positioned inrelation to the holder to punch a tissue specimen from the tissue donorblock when the donor block is in the donor position; and a recipientblock holder for holding a recipient block in a recipient position,wherein the recipient block comprises an array of receptacles, each ofwhich is positionable in a preselected position in relation to the itreciprocating punch to deliver a tissue specimen from the firstreciprocating punch into a receptacle in the preselected position; and asecond reciprocating punch capable of being positioned relative to therecipient block for punching the array of receptacles in the recipientblock, wherein the second reciprocating punch is different than thefirst reciprocating punch positioned to punch the specimen from thetissue donor block; and a reference slide positioner that includes atleast one slide that extends between opposing walls of the donor blockholder.
 14. A device for preparing biological material arrays,comprising: a platform that includes at least one guide for positioninga tissue donor block holder or a recipient block holder; and a punchapparatus that includes it guide surface, a punch base slidably mountedon the guide surface, an a punch received within the punch base that canhe aligned with the tissue block holder or the recipient block holder;and a reference slide positioner interposed between the platform and thepunch apparatus.
 15. The device of claim 14, further comprising meansfor sliding the punch base.
 16. An integrated apparatus for preparingspecimens for parallel analysis of sections of biological materialarrays, comprising: a donor block holder that can hold a tissue donorblock in a donor position; a first reciprocal punch positioned inrelation to the donor block holder that can punch a tissue specimen fromthe tissue donor block when the donor block is in the donor position; arecipient block holder that can hold a recipient block in a recipientposition, wherein the recipient block comprises an array or receptacles,each of which is positionable in a preselected position in relation tothe first reciprocal punch to deliver a tissue specimen from the firstreciprocal punch into a receptacle in the preselected position; and asecond reciprocal punch capable of being positioned relative to therecipient block for punching the array of receptacles in the recipientblock, wherein the second reciprocal punch is different than the firstreciprocal punch positioned to punch the specimen from the tissue donorblock; and a positioner that can position over the donor block areference slide that includes at least one structure of interest toalign the at least one structure of interest in the reference slide withcorresponding tissue specimen regions in the donor block.
 17. Theapparatus of claim 16, further comprising z-direction positioning meansfor the first reciprocal punch and the second reciprocal punch.
 18. Theapparatus of claim 16, wherein the diameter of the first reciprocatingpunch positioned to punch the specimen from the tissue donor block isgreater than the diameter of the second reciprocating punch.
 19. Anapparatus for preparing specimens for parallel analysis of sections ofbiological material arrays, comprising: an x-y positioning platform; adonor block holder for holding a tissue donor block in a donor position,the donor block holder being disposed on the x-y positioning platform; areciprocating punch positioned in relation to the donor block holder topunch a tissue specimen from the tissue donor block when the donor blockis in the donor position; a recipient block holder for holding arecipient block in a recipient position, wherein the recipient blockcomprises an array of receptacles, each of which is positionable in apreselected position in relation to the reciprocating punch to deliver atissue specimen from the reciprocating punch into a receptacle in thepreselected position, the recipient block holder being disposed on thex-y positioning platform; a sectioning device for sectioning therecipient block into sections that can be subjected to differentanalyses; and z-direction positioning means for the reciprocating punch.20. An apparatus for preparing specimens for parallel analysis ofsections of biological material arrays, comprising: an x-y positioningplatform; a donor block holder for holding a tissue donor block in adonor position, the donor block holder being disposed on the x-ypositioning platform; a reciprocating punch positioned in relation tothe donor block holder to punch a tissue specimen from the tissue donorblock when the donor block is in the donor position; a recipient blockholder for holding a recipient block in a recipient position, whereinthe recipient block comprises an array of receptacles, each of which ispositionable in a preselected position in relation to the reciprocatingpunch to deliver a tissue specimen from the reciprocating punch into areceptacle in the preselected position, the recipient block holder beingdisposed on the x-y positioning platform; a recorder for recordingcoordinate positions of the receptacles in the recipient block; andz-direction positioning means for the reciprocating punch.